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BOSTON UNIVERSITY SCHOOL of MEDICINE Dissertation BOSTON UNIVERSITY SCHOOL OF MEDICINE Dissertation DEAMIDATION AND RELATED PROBLEMS IN STRUCTURAL ANALYSIS OF PEPTIDES AND PROTEINS by NADEZDA P. SARGAEVA B.S., St. Petersburg State Polytechnical University, 2002 M.S., St. Petersburg State Polytechnical University, 2004 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2012 Approved by First Reader Peter B. O’Connor, Ph.D. Associate Professor of Biochemistry Second Reader Cheng Lin, Ph.D. Assistant Professor of Biochemistry Dedications I dedicate this thesis to my Family iii Acknowledgments I would like to express my deepest gratitude to my advisor, Prof. Peter B. O’Connor. We met in St. Petersburg, Russia in 2004 and you believed in my abilities and you gave me an opportunity to be a part of such a great mass spectrometry program. I also want to thank you for your support and your guidance throughout my Ph.D. studies. Thank you for pushing me to achieve my goals of learning and becoming a better scientist, and for the opportunity you gave me to build up my professional and scientific network. Your help, your advice, and your encouragement in building up my professional career are very much appreciated. Prof. Cheng Lin, thank you for being my second reader and mentor. Working with you has always been a pleasure – I highly value your well thought out ideas, your constant use of the scientific principle, and your willingness to follow up regarding the data, the future directions, etc. I truly appreciate your attention to details, your regular availability and willingness to help me hands on. Thank you for mentoring me not just throughout the program but also regarding the life journey I’m about to embark on. Peter and Cheng, you are the two most influential people that helped me achieve my goals and I thank you very much from the bottom of my heart. I would like to express my gratitude to the other members of my thesis advisory committee: Prof. Carmela Abraham, Prof. Matthew Nugent, Prof. Mark E. McComb, and Prof. Joseph Zaia for spending your valuable time serving on my committee and giving me constructive criticism and advice. Special thanks to Mark (along with Nancy Leymarie), for spending a lot of time with me working on the last challenging project – your contribution is very much appreciated. iv I am sincerely thankful to Prof. Catherine E. Costello. Your wealth of experience and, more importantly your willingness to share it, have been very important to my development. Thank you for your valuable advice and your contribution to my studies. The research groups of Prof. Dieter Seebach, Prof. Sam Gellman, Prof. Kaj Blennow, and Prof. Mikhail Gorshkov are highly appreciated for their great collaborations and inimitable samples. My dear fellow former and present students and colleagues, it was a pleasure to get to know you and work with you. Special gratitude goes to Dr. Chunxiang Yao, Dr. Weidong Cui, Amanuel Kehasse, Dr. Nancy Leymarie, and Dr. Sandrine Voillard Bourgoin. Dr. Jason Cournoyer, Dr. Cheng Zhao, Dr. Vera Ivleva, and Dr. David Perlman thank you for your immense help during my initial stages of the program. Dr. Konstantin Aizikov, Prof. Alexander Ivanov, Dr. Bogdan Budnik, and Prof. Eugene Moskovets, thank you for your friendship and your willingness in help through all the years of my Ph.D. studies. Eugene, I truly appreciate your help well before and throughout the program; you are always ready with scientific advice and life guidance. Most importantly, my wonderful family and friends, I’m so blessed to have all of you in my life. My beloved husband, Alexander Cherkassky, I would not be able to go through the obstacles, especially during the last months, without your endless help, moral support, and infinite love. I’m very lucky to deserve all the kindness and unconditional love of your wonderful family, especially, your Mother, Polina, and your sister, Rimma. To the best possible parents in life, my Mother, Alla, and my Father, Pavel, and to my dearest sister, Olga - I never feel that we are across the ocean; you are always here with me in my heart. I can’t be thankful enough for you being there for me at all times – the day or the night. v DEAMIDATION AND RELATED PROBLEMS IN STRUCTURAL ANALYSIS OF PEPTIDES AND PROTEINS (Order No. ) NADEZDA P. SARGAEVA Boston University School of Medicine, 2012 Major Professor: Peter B. O’Connor, Ph.D., Associate Professor of Biochemistry ABSTRACT Electron capture dissociation (ECD) and electron transfer dissociation (ETD) can generate unique fragments and preserve post-translational modifications (PTMs), enabling their detection in biological samples. They have been used to differentiate isomeric aspartic (Asp) and isoaspartic acids (isoAsp) produced upon non-enzymatic deamidation of asparagine (Asn) – a frequently occurring PTM. IsoAsp formation was detected in amyloid-β (A β) peptides in the specimens of Alzheimer’s disease (AD) patients, and is a potential biomarker for AD if it can be detected early in biofluids of live individuals. Synthetic isoAsp-containing A β fragments were studied using ECD to test the method's applicability. IsoAsp-7 and -23 were detected in top-down analysis of the 4.5kDa A β42 protein and in A β17-28 peptide. Further, a related method, electron ionization/impact dissociation (EID), was successfully applied to Asp/isoAsp differentiation for the first time. High-performance liquid chromatography (HPLC) is a powerful technique for the separation of complex mixtures. HPLC separation of Asp- and isoAsp-containing peptides revealed inconsistent elution orders, especially when isoAsp was located at the N-terminus, requiring ECD for identification. New diagnostic vi fragments were proposed for N-terminal isoAsp based on the ECD and ETD results. Challenges in detection of such fragments were improved by supplemental activation and chemical modifications. Furthermore, a model for retention time prediction was applied to isoAsp-containing peptides and suggested for their improved identification in HPLC-MS/MS approach. IsoAsp is a β-amino acid, which distinctively contains an additional methylene group in the backbone, forming a Cα-Cβ bond. Cleavage of this bond provides diagnostic fragments for isoAsp by ECD. The same was proposed for other β-amino acids. However, the Cα-Cβ bond cleavages were rare due to the instability of the C β radical. Alternatively, in-source decay (ISD) fragmentation during matrix- assisted laser desorption/ionization (MALDI) process can produce abundant ECD-like fragmentation. It was proposed that use of hydrogen-accepting matrices may lead to Cα-Cβ bond cleavage in β-amino acids, because the resulting radical would be stabilized by the carbonyl group. To test this, β-amino acid-containing peptides were analyzed by MALDI-ISD using 5-nitrosalicylic acid matrix. The Cα-Cβ bond cleavages were observed. Overall, new and improved methods were implemented allowing better characterization and differentiation of β-amino acids. vii Table of contents Title page…………………………………………………………………………………………..i Reader’s approval page…………………………………………………………………………ii Dedications……......……………………………………………………………………………. iii Acknowledgements..…………………………………………………………………………….iv Abstract…………………………………………………………………………………………...vi Table of contents ............................................................................................................. viii List of Tables .................................................................................................................. xiv List of Figures .................................................................................................................. xv List of Abbreviations ........................................................................................................ xx Chapter 1 1 Introduction 1 1.1 Introduction to Mass Spectrometry in the Analysis of Biomolecules ........................... 1 1.2 Introduction to Mass Spectrometry ............................................................................. 2 1.2.1 Ionization Techniques and Ionization Sources .................................................... 3 1.2.2 Mass Analyzers ................................................................................................. 10 1.2.3 Tandem Mass Spectrometry ............................................................................. 29 1.2.4 ESI QqQ-FTICR mass spectrometer ................................................................. 37 1.2.5 Bruker Daltonics solariX FTICR MS .................................................................. 40 1.3 High Performance Liquid Chromatography (HPLC) .................................................. 42 1.3.1 Reversed Phase High Performance Liquid Chromatography (RP-HPLC) ........ 44 1.3.2 On-line and off-line Liquid Chromatography Mass Spectrometry ...................... 48 1.4 Introduction to deamidation and isomerization of peptides and proteins .................. 49 viii 1.4.1 Deamidation and isomerization of peptides and proteins .................................. 49 1.4.2 Biological importance of deamidation and isomerization ................................... 51 1.5 Introduction to β-peptides .......................................................................................... 53 1.6 Thesis overview ........................................................................................................ 56 Chapter 2 57 Identification of isoAsp in Amyloid βββ Peptides by Tandem ExD MS/MS 57 2.1 Introduction ..............................................................................................................
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